Volume 72, Issue 4, Pages 422-429 (August 2007) Organic cation transport in the rat kidney in vivo visualized by time-resolved two-photon microscopy  M. Hörbelt, C. Wotzlaw, T.A. Sutton, B.A. Molitoris, T. Philipp, A. Kribben, J. Fandrey, F. Pietruck  Kidney International  Volume 72, Issue 4, Pages 422-429 (August 2007) DOI: 10.1038/sj.ki.5002317 Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 1 Short-term distribution of fluorescence in the kidney after ASP+ injection. A 0.2 ml bolus containing 0.3 μmol ASP+ (1.5 mM final concentration) was injected intravenously and kidney fluorescence was continuously monitored by 2P-LSM. (a) Before injection, only autofluorescence was observed in proximal tubule segments. (b and c) After ASP+ injection, fluorescence appeared in peritubular capillaries omitting red blood cells (×). In parallel, fluorescence appeared outside the capillaries and a transient, pronounced, and blue-shifted fluorescence was observed at basolateral membranes (arrows). Subsequently, intracellular fluorescence rapidly increased in proximal and distal (asterisk) tubules starting from the basolateral side, omitting the nuclei. (d–f) A transient and pronounced fluorescence was observed at the brush-border membranes of proximal tubules where it was shifted to shorter wavelengths (arrowheads). (g–i) Intracellular fluorescence decreased slowly over time. The length of the sizing bar corresponds to 50 μm. Kidney International 2007 72, 422-429DOI: (10.1038/sj.ki.5002317) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 2 Prolonged distribution of fluorescence in the kidney after ASP+ injection. A 0.2 ml bolus containing 0.3 μmol ASP+ (1.5 mM final concentration) was injected intravenously. Subsequently, ASP+ distribution in the kidney cortex was monitored over 40 min by 2P-LSM. (a) Capillary fluorescence omitted red blood cells (×). Transient and pronounced fluorescence was observed at basolateral (arrows) and at brush-border membranes (arrowhead) where it was shifted to shorter wavelengths. Intracellular fluorescence in proximal and distal (asterisk) tubules omitted the nuclei. (b–d) Intracellular fluorescence of proximal tubule segments decreased to baseline within 40 min. During this time, intracellular fluorescence in distal tubules (asterisks) was largely preserved and was partially translocated into subcellular vesicles at the apical aspect of these cells (circles). The length of the sizing bar corresponds to 50 μm. Kidney International 2007 72, 422-429DOI: (10.1038/sj.ki.5002317) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 3 Effect of cimetidine on tubular ASP+ transport. Fluorescence intensity of proximal tubules, continuously recorded by 2P-LSM after injection of ASP+ only and ASP+ together with cimetidine. Depicted are two typical experiments. (a) In the first experiment, an ASP+ control injection was followed by an injection of ASP+ plus cimetidine. (b) In the second experiment, the first injection contained ASP+ together with cimetidine and was followed by a control injection containing ASP+ alone. Each 0.2 ml injection bolus contained either 0.3 μmoles ASP+ alone (1.5 mM final concentration) or 0.3 μmoles ASP+ plus 8 μmoles cimetidine (40 mM final concentration), respectively. The second injection was started 35 min after the first injection as indicated. Kidney International 2007 72, 422-429DOI: (10.1038/sj.ki.5002317) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 4 Changes of fluorescence caused by the filtered ASP+ fraction. A low molecular weight fluorescent dextran (3 kDa) labeled with Texas Red was utilized to visualize the luminal passage of the glomerulus filtrate. ASP+ and dextran were co-injected into the jugular vein and fluorescence of the kidney cortex was continuously monitored by 2P-LSM. (a–c) Shortly after injection, fluorescence increased in the peritubular capillaries (×). (c–j) In an early proximal tubule segment (d, 1), transient luminal red fluorescence, supposedly originating from filtered Texas Red, occurred 7.5 s after injection. This was paralleled by a transient and pronounced green–yellow fluorescence at the brush border originating from filtered ASP+ (arrowhead in e). (k–o) In a late proximal tubule segment (l, 2), the transient luminal fluorescence of Texas Red occurred 18.75 s after injection. This was not paralleled by brush-border fluorescence (arrowhead in m). The tubule segment with early luminal fluorescence and intense brush-border fluorescence subsequently exhibited higher intracellular fluorescence than the tubule segment with late luminal fluorescence (o). The length of the sizing bar corresponds to 25 μm. Kidney International 2007 72, 422-429DOI: (10.1038/sj.ki.5002317) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 5 Segmental differences of proximal tubule fluorescence as a function of time after ASP+ injection. (a–c) ASP+ was injected intravenously and fluorescence of the kidney cortex was continuously monitored with 2P-LSM. Images (a and b) display the ASP+ fluorescence of proximal tubule segments at 15 and 35 s after injection, respectively. Panel c displays the maximal intensity determined for each pixel over 45 s as a composite image. In all proximal tubule segments in the area of view, a transient and pronounced ASP+ fluorescence occurred at the brush-border region within 45 s, shifted to shorter wavelengths. The numbering (1–6) of the imaged tubule segments denotes the sequence of the transient brush-border fluorescence. The ‘×’ denotes the lumen of a peritubular capillary. The sizing bar corresponds to a distance of 50 μm. (d–f) The graphs display fluorescence changes in a peritubular capillary (d ‘ × ’), in the intracellular part (e) and the brush-border regions (f) of different proximal tubule segments (1–6) over time. Numbering in graphs (d–f) corresponds to numbered tubule segments (a–c). Shortly after the capillary fluorescence (d ‘×’), intracellular fluorescence increased in all tubule segments (e). Brush-border fluorescence intensity inversely correlated with the time of appearance (f). The tubule segment with earliest and most intense brush-border fluorescence (f ‘①’) subsequently exhibited a higher intracellular fluorescence (e ‘①’) than other segments. Kidney International 2007 72, 422-429DOI: (10.1038/sj.ki.5002317) Copyright © 2007 International Society of Nephrology Terms and Conditions